The overall goal of this project is to understand how papillomaviruses replicate their genomes. Papillomaviruses have in recent years emerged as very important causal agents in human disease. These viruses, as a part of their normal life cycle, infect and transform cells in the epithelium causing benign tumors that with a low, but significant, frequency can become malignant. A deeper understanding of the viral life cycle in general, and DNA replication in particular, is of critical importance for the understanding of the disease, its transmission, and ultimately for the development of effective therapeutic measures. We are studying DNA replication of papillomaviruses in vivo and in vitro. We are combining genetic biochemical and structural analyses of the viral proteins and DNA sequence elements that are required for viral DNA replication. In this project we will continue our in depth analysis of the papillomavirus replicon with emphasis on the assembly pathways responsible for the ordered recognition, distortion and unwinding of the viral origin of replication. We propose (I) to analyze the formation of the E1E2-ori complex and its DNA binding; (ii) to characterize the DNA binding activity of the full-length E1 protein; (iii) to define the ori distortion activity of E1; and finally (iv) to analyze the assembly and properties of the hexameric E1 helicase. These studies will further our understanding of how papillomavirus replication is initiated and controlled. In addition, the elucidation of these processes at the molecular level will result in novel targets for drug intervention.